Genetic hemochromatosis is a prevalent disorder of iron balance. The most common form of the disease results from mutations in HFE, an MHC class I protein of uncertain function. The goal of this proposal is to understand where and how the HFE protein normally functions to regulate iron homeostasis. To fully understand HFE, we feel that it is necessary to study iron homeostasis in living animals. We have taken a genetic approach to this problem, using transgenic mouse technology to alter individual components of iron transport and regulatory pathways, to determine how perturbation of one step alters iron balance. This application describes experiments for that purpose, and focuses on a new hypothesis: that HFE normally serves to modulate expression of hepcidin, a peptide hormone produced by the liver, and that hepcidin acts to attenuate release of iron from absorptive enterocytes and iron-recycling macrophages. In Aim 1, we will attempt to identify the cellular site of Hfe activity in vivo, by characterizing mice lacking the protein selectively in macrophages, hepatocytes or enterocytes. In Aim 2 we will investigate the importance of the interaction between Hfe and the transferrin receptor in vivo. In Aim 3, we will determine whether HFE is only involved in regulation of iron absorption in response to body iron stores, or whether it also participates in regulation in response to erythropoietic drive, hypoxia and inflammation. Finally, in Aim 4 we will explore the current model that hepcidin acts to attenuate release of iron from intestinal absorptive cells and macrophages. Together, these experiments should help to define the HFE/hepcidin regulatory axis, and inform future investigations of the pathogenesis of hemochromatosis.